The present invention relates to the sequence of genes from the E. canis bacterium, and the development of a vaccine against this organism.
Ehrlichia canis (E. canis) is a small gram-negative, obligately intracytoplasmic rickettsia. This bacteria is the agent which causes canine monocytic ehrlichiosis (CME), a tick-borne disease which predominantly affects dogs. The most common carrier of E. canis is the brown dog tick Rhipicephalus sanguineus. The disease was described originally in Algeria in 1935. It was subsequently recognized in the United States in 1962, but is now known throughout much of the world. Canine monocytic ehrlichiosis caused much concern during the Vietnam War, when 160 military dogs died from the E. canis infection. There is no vaccination currently available against E. canis. It is a life threatening disease that continues to be an important health concern for veterinarians and pet owners alike.
Canine monocytic ehrlichiosis is an infectious blood disease. A reduction in cellular blood elements is the primary characteristic of the disease. E. canis lives and reproduces in the white blood cells (leukocytes). It eventually affects the entire lymphatic system, and devastates multiple organs. By targeting the white blood cells, these cells die off rapidly. These dead blood cells migrate primarily to the spleen, which enlarges as a result. The bone marrow recognizes the loss of the white blood cells and works to form new, healthy cells. It sends out the cells prematurely, and these immature cells do not work properly. Often, these immature cells mimic those in leukemic patients, so the disease is misdiagnosed as leukemia. Canine monocytic ehrlichiosis may also predispose dogs to various cancers.
There are three stages of canine monocytic ehrlichiosis. The first, acute stage mimics a mild viral infection. During the acute stage, most, if not all, of the damage is reversible and the animal is likely to recover. This is the stage where treatment is the most effective, stressing the need for early detection. Without treatment, however, the animal will progress into a subclinical (second) stage and/or to the chronic (final) stage. When the animal has reached the chronic stage, the bacterial organism has settled within the bone marrow. Many dogs in this stage suffer massive internal hemorrhage, or develop lethal complications such as sudden stroke, heart attack, renal failure, splenic rupture or liver failure.
E. canis can be cultured in vitro in a mammalian-derived cell line (DH82). Continued maintenance of these cells is difficult because the cell culture must be supplemented with primary monocytes (white blood cells found in bone marrow) every two weeks. The cultures are very slow growing, and the culture media is expensive.
Data concerning the genes in the E. canis genome has concentrated primarily on the 16S rRNA gene. Previous work has sequenced this gene, which is a ubiquitous component of the members of the ehrlichia family, as well as the majority of organisms worldwide. The high sequence homology between this gene throughout the living world and its poor immunogenicity makes it an unsuitable candidate for vaccine development. It is necessary to find other genes within this genome if hope for a vaccine against this deadly disease can ever be realized.
Sequencing of the 16S rRNA gene indicates that E. canis is closely related (98.2% homology) to E. chaffeensis, the novel etiologic agent of human ehrlichiosis. Western blots of E. canis are similar when probed with antisera to E. canis, E. chaffeensis, and E. ewingi (another cause of human ehrlichiosis) indicating a close antigenic relationship between these three species (Chen et al., 1994).
The indirect fluorescent antibody test (IFA) has been developed for detecting canine monocytic ehrlichiosis. IFA detects the presence of antibodies against the invading organism in a dog's blood. Unfortunately, this test is not always accurate. Sometimes, dogs will test negative in the acute phase because their immune system is delayed in forming antibodies. Another false negative may occur if there is a low titer in the chronic stage. An additional drawback of this test is the cross-reactivity found. The anti E. canis polyclonal antibody positively reacts with E. chaffeensis, undermining the specificity of the test. An alternative test, the Giesma smear, has been used to locate the actual organism in a dog's blood. Unfortunately, despite appropriate staining techniques and intensive film examination, the organisms frequently can not be located. The fallibility of these tests makes it essential to provide better diagnostic tools for this disease.
Due to difficulties in the detection of a tick bite, early diagnosis of infection, the suppression of host defenses and the nature of persistent infection of the disease, an effective vaccine against E. canis is urgently needed for dogs.